The present invention is directed, in general, to a method for the identification of the molecular targets for drugs or toxins in an organism or other biological system.
Most drugs or toxins express their activity by binding to proteins. These proteins are referred to as receptors, drug targets or molecular targets (Gies, 1996). Drugs (pharmaceuticals), toxins and other biologically active molecules will be referred to herein as ligands. Identification of the ligand target is the crucial first step in understanding how a ligand affects a biological system. Currently, this identification is usually a long and arduous process. The identification of a ligand's target is desirable, however, because it provides essential information for the improvement of the drug or assessment of toxicity or side effects.
Many drugs are now designed specifically to bind to a particular target protein, and their primary target is not in doubt. However, it is possible for these drugs to have additional targets to which they bind that give rise to unexpected or unwanted biological effects (toxicities or side effects). The origin of these side effects or toxicities is not always clear from the primary mode of action of the drug. Identification of secondary targets, the interaction with which leads to side effects, may aid initial toxicological evaluations on humans by identifying potential biological systems to monitor, aiding in the interpretation of observed adverse effects, or providing information that could be used to counteract these effects.
In addition to designed drugs, natural products or synthetic organics are often screened for a particular biological activity (e.g., killing of human cancer cells in culture), and those displaying desirable activities are identified and developed without foreknowledge of the molecular target through which its activity is derived. The first step in understanding the mode of action of these drugs is to determine the molecular target of the drug. This is often a slow and expensive process. However, identification of the primary and secondary targets of these types of drugs is crucial to their further development and toxicological evaluations.
Prior to human testing of a new drug, a drug is tested on animals to evaluate its toxicity. The success of these toxicological screens depends on the efficacy with which the animal model mimics the human systems to be effected. If the molecular targets of the animal are essentially identical to those of humans, the toxicological evaluation in an animal will be an accurate guide to the toxicity of the drug in humans. This is, however, not universally true. Many drugs and toxins are highly species dependent in their action (for instance, aspirin is toxic to mice, Ohdo, et al., 1995). If a list of potential human molecular targets were available prior to testing in animals, one could choose a more appropriate test animal. For instance, if one potential target of a drug is the enzyme hexokinase (the first enzyme in the glycolytic pathway), the sequences of human and mouse hexokinase could be compared; if these sequences are similar at the postulated drug binding site then a mouse is an acceptable model for the evaluation of the effect of a drug on glycolysis; if not, then use of another animal model would be indicated. Consequently, the ability to predict potential drug binding sites in advance of animal testing would aid in the design and evaluation of toxicological screens. Furthermore, during clinical trials, a list of potential targets would simplify the evaluation of adverse effects of the drug.
There have been instances during the clinical use of a drug where unexpected benefits have been observed, identifying a drug being used to treat one pathology as efficacious against another one. This is particularly advantageous since a drug in clinical use has already passed through many regulatory hurdles and completed toxicological evaluations. A list of potential targets for a drug already in use could provide clues to new applications of the drug, and provide lists of pathologies against which the drug should be tested. This would be particularly beneficial for rare diseases where there is little financial incentive for drug development.
In addition to the determination of the mode of the desired interactions of pharmaceuticals, the identification of molecular targets is also essential in understanding the effects of environmental toxins. Man-made and naturally occurring toxins present a continual danger to human populations. Assessment of the risks posed by these molecules is dependent on determining the mode of action of the toxins. Further, these molecules may, in some cases, have several independent physiologically important targets. A complete characterization of the risk associated with exposure to these toxins involves identification and characterization of all relevant molecular targets.
Current methods of identifying the molecular targets of drugs or toxins in biological systems are cumbersome. Usually, they involve culturing large amounts of mammalian or other organismal cells in order to harvest enough protein extract to test for binding to the ligand of interest. Once these proteins are extracted, they must be isolated in sufficient amounts for protein sequencing by affinity to the ligand (a difficult task for low-expression proteins). Then, the purified protein must be partially sequenced by Edman degradation and a putative peptide sequence determined. If this sequence is of sufficient quality, then a set of degenerate DNA hybridization probes must be devised to screen the genomic library of the original cell of interest. If this process is successful, then the gene for the protein may be recovered, cloned into an expression vector, and later sequenced. Although this process will yield the identity of the protein suspected of binding to the ligand, the steps of cell culturing, purification, peptide sequencing, and probing for hybridization of the gene of interest, are all costly and time consuming.
Sparks et al. (1996) and Hoffmann et al. (1996) reported that they screened human and mouse protein libraries generated from cDNA to identify proteins with high affinity for specific peptides. They have described only screens against peptides (not, e.g., small molecule drugs or toxins). Also, random peptide libraries are sold commercially for screening against antibodies to identify epitopes (New England Biolabs Product Catalog, “Ph.D” products, 1998), another form of protein—protein interaction. Screening for proteins that demonstrate high affinity for peptide ligands is both conceptually and practically different from using small molecules as ligands. Protein-protein interactions generally involve the spacio-chemical interaction of large structures on each protein, generally encompassing relatively large sites of interaction. Thus, binding energy is ordinarily much stronger for peptide ligands. However, the applications of this technique are relatively limited, as many biologically active molecules of interest are not proteinaceous.